CN102936620B - Method for detecting nucleotide sequence by using poly(methyl acrylic pyrene-poly (methyl) acrylic dimethylamine ethyl ester copolymer, and product thereof - Google Patents
Method for detecting nucleotide sequence by using poly(methyl acrylic pyrene-poly (methyl) acrylic dimethylamine ethyl ester copolymer, and product thereof Download PDFInfo
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Abstract
The invention relates to a method for detecting nucleotide sequence by using poly(methyl acrylic pyrene-poly (methyl) acrylic dimethylamine ethyl ester copolymer, and a product thereof, and belongs to the field of functional polymers. The method comprises the steps of protonating the poly(methyl acrylic pyrene-poly (methyl) acrylic dimethylamine ethyl ester copolymer with alkyl halide to obtain a polyelectrolyte having positive charges, and acting the polyelectrolyte with a linear or a hairpin structure DNA to form a novel fluorescent probe for detecting DNA. The method is advantageous in that the product is the novel fluorescent probe which is simple in preparation, easy to operate and effective, provides a novel approach for stable, efficient and specific identification of the nucleic acid molecules, and has significant scientific and application value in aspects of disclosing diseases and genetic variations and the like.
Description
Technical field
One utilizes polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer to detect the method for nucleotide sequence, particularly relate to a kind of DNA of non-conjugated fluorescent copolymer and straight chain or hairpin structure that utilizes and form by electrostatic interaction the method that novel molecular probe detects target dna, belong to functional polymer technical field.
Background technology
The functional polymer application in biomedical sector, particularly fluorescence polymer are in the close attention analyzed, the research detected in biomacromolecule has caused domestic and international scientific worker, and this research direction is also the study hotspot of front line science in the world.Some small molecule fluorescent dyestuffs such as acridine, phenanthridines class dyestuff, cyanine type dye, fluorescein and dye stuff of rhodamine kinds, Sai Qin He oxazine class dyestuff etc. are the nucleic acid fluorescent probes that application is wider at present, these simple fluorescence dyes can not specific recognition to the sequence of nucleic acid molecule, belongs to nonspecific Small-molecule probe.In order to overcome above-mentioned deficiency, fluorophor is attached on strand nucleic acid oligomer molecule by covalent linkage by scientific worker, such as: the people such as Saito, Hrdlicka devise a kind of pyrene respectively and mark linear fluorescence probe on nucleic acid, find that the nucleic acid molecule of this fluorescently-labeled molecular probe and base sequence complementary forms the multiple chain of hydridization by Watson-Crick base pairing rules, fluorescence property generation significant change before and after target hydridization, thus achieve fluorescent probe specific recognition nucleic acid molecule (J.Am.Chem.Soc., 2004,126,4820; Chem.Commun., 2010,46,4929).The people such as Wang also devise a kind of pyrene mark HNA and RNA linear probe, after finding target hydridization, the fluorescence intensity of pyrene monomer increases and the fluorescence intensity of exciplex acutely declines, thus realizes detection (ChemBioChem., 2009 to target molecule, 10,1175).But relative to linear molecular probe, it is the molecular probe of the hairpin structure that a kind of stem end has a pair fluorophor and cancellation group to modify, also referred to as molecular beacon, there is the advantage of some keys: improve the heat identification of mismatched target and reduce the risk of false positive signal.Thus be subject to paying close attention to more widely at biological research fields.The people such as Tan have made a lot of outstanding achievement in the design and application of molecular beacon, comprise monitoring etc. (J.Am.Chem.Soc., 2008,130,8351. of the design of biosensor, the detection of nucleic acid and Living system; Angew.Chem., 2001,113,416; Angew.Chem.Int.Ed., 2001,40,402.; Anal.Chem., 2005,77,4713).But result of study shows: the shortcoming that marking type fluorescent probe detects nucleic acid molecule is: the introducing of fluorophor can reduce the thermostability of fluorescent probe and targeted molecular heteroduplex, thus can reduce the accuracy of detection of fluorescent probe; Marking type fluorescent probe needs fluorophor to be attached to realize specific recognition nucleic acid molecule on nucleic acid oligomer molecular chain by covalent linkage, and synthesis is wherein more numerous and diverse with operation.
In recent years, the parent that water miscible fluorescence conjugated polyelectrolyte obtained increasing investigator as the fluorescent probe fluorescence of non-marking in bio-sensing Application Areas looks at.When the multiplexed DNA probe that the people such as He report non-marked type prepared by a kind of conjugated polyelectrolytes is to detect DNA hybridization, signal transfers (J.Am.Chem.Soc., 2009,131,3432).The people such as Bazan are by the fluorescent probe (Chem.Mater., 2004,16,4467) with a kind of water miscible positively charged ion conjugated polyelectrolytes design homogeneous.The people such as Wang Shu also report a kind of molecular beacon that makes full use of in the advantage of mismatch binding and the higher character of quantum yield of conjugated polyelectrolytes and the molecular probe of the detection DNA prepared, and the fluorescence micella that there was reported with including conjugated polyelectrolytes detects DNA and enzyme etc. (Langmuir., 2008,24,12138; Langmuir., 2009,25,13737; Angew.Chem.Int.Ed., 2009,48,5316).These fluorescence conjugated polyelectrolyte are combined with biomacromolecule by electrostatic interaction, the optical property of this fluorescence polymer can be caused to change by FRET (fluorescence resonance energy transfer) FRET or transfer transport, the change of analyte induced fluorescence macromolecule aggregating state structure, analyte induced fluorescence polymer conformational change etc., and then biomacromolecule is detected.Although conjugated polyelectrolytes has the material being applied as biosensor widely, there is not been reported as biosensor material for non-conjugated fluorescence polyelectrolyte.A kind of cold fluorescence polyelectrolyte newly of invention, it is formed a kind of novel cold fluorescent probe by electrostatic interaction and DNA, this fluorescent polymer overcomes the shortcomings such as the preparation of the non-specific identification of small-molecule fluorescent probe and markup fluorescent probe is numerous and diverse, for the macromolecular detection of biology opens a kind of new testing method.
The patent name of applicant's application is a kind of method utilizing vinylformic acid pyrene methyl esters to prepare water solubility copolymer, application number: the patent application of 201110430560.7, disclose not protonated before the synthetic method of polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer, the application quotes in full at this.
Summary of the invention
The object of the present invention is to provide a kind of synthetic method simple, and the non-conjugated polyelectrolyte containing the higher pyrene fluorophor of fluorescence quantum yield, form in conjunction with the DNA of straight chain or hairpin structure the method that novel molecular probe detects target dna by electrostatic interaction.
Feature of the present invention is to utilize non-conjugated polyelectrolyte to be combined with DNA, form novel molecular probe, this non-conjugated polyelectrolyte synthetic route is simple, the side chain of synthesized polymkeric substance introduces fluorescence quantum yield higher and have the pyrene fluorophor of very strong susceptibility and hydrophilic amine groups to surrounding environment, make this polyelectrolyte can be advantageously applied to the detection of the aspects such as biological.
The object of the invention is to be achieved through the following technical solutions: the present invention utilizes polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer to make the positively charged polyelectrolyte containing pyrene fluorophor through haloalkane is protonated, and the single stranded DNA effect of this polyelectrolyte and straight chain or hairpin structure is formed novel fluorescence probe detect target dna.
A kind of method detecting nucleotide sequence with polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer, carry out according to following step:
(1) reaction that multipolymer is protonated:
Be that 1:3 is dissolved in organic solvent tetrahydrofuran by polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer and appropriate methyl iodide by amount of substance, react 24 hours at normal temperatures.After reaction terminates, solvents tetrahydrofurane is filtered out, and then extracts product with apparatus,Soxhlet's, wherein extraction agent is tetrahydrofuran (THF), extraction time is 12 hours, finally again by final fluorescence polyelectrolyte in vacuum drying oven with dry 12 hours of 70 DEG C of temperature, synthetic route is as shown in Figure 1.
(2) preparation of Novel fluorescent molecular probe:
Use buffered soln PBS(10Mm, pH=7.4) original solution is made in polyelectrolyte dilution obtained in (1), equally, use buffered soln PBS(10Mm respectively, pH=7.4) single stranded DNA 1 of linear chain structure or the single stranded DNA 2 of hairpin structure are dissolved, finally, then get suitable polyelectrolyte solution and mix with DNA1 or DNA2 solution, by electrostatic interaction capable become the fluorescent molecular probe of novel straight chain or hairpin structure.
(3) preparation of DNA hybridization sample:
Use buffered soln PBS(10Mm, pH=7.4) dilution not homotactic DNA3, DNAa, DNAc, DNAt.Wherein DNA3 and DNA1 complete complementary pairing, completely not complementary with DNAa, DNAc, DNAt; DNA1 and DNA2 partial complementarity matches, completely not complementary with DNAa, DNAc, DNAt.Get the DNA3 that a certain amount of dissolving is good respectively, in the fluorescent probe sample solution prepared during DNAa, DNAc, DNAt add (2), finally by the thermal treatment of 90 DEG C through 5min of all solution of preparing, then be incubated half an hour at 40 DEG C.
The described polyacrylic acid pyrene methyl esters synthesized by step (1)-poly-(methyl) vinylformic acid dimethylamine ethyl ester polyelectrolyte structure is:
Wherein x/y=1:10 ~ 1:400.R=CH
3,CH
2CH
3,CH
2CH
2CH
3,CH
2CH
2CH
2CH
3；X=I,Br。
Containing fluorophor pyrene in the described polyacrylic acid pyrene methyl esters synthesized by step (1)-poly-(methyl) vinylformic acid dimethylamine ethyl ester polyelectrolyte, and except fluorophor pyrene, other fluorophors as: the fluorophors such as green onion, anthracene, naphthalene are also passable.
Described step (1) polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer and methyl iodide are dissolved in organic solvent tetrahydrofuran, and multipolymer is protonated, wherein the ratio 1:1 ~ 1:10 of fluorescent copolymer and the amount of methyl iodide.
Described step (2) and (3) buffered soln used are PBS(10Mm, pH=7.4), DNA used is respectively: DNA1:5 '-GCA CAT ACA TTC TAC TTG-3 '; DNA2:5'-GCACAAACAAGTAGAATGTATGTGC-3 '; DNA3:5 '-CGTGTA TGT AAG ATG AAC-3 '; DNAa:5 '-AAA AAA AAA AAAAAAAAA-3 '; DNAc:5 '-CCC CCC CCC CCC CCC CCC-3 '; DNAt:5 '-TTTTTT TTT TTT TTT TTT-3 ', wherein DNA2 is hairpin structure, and DNA1, DNA3, DNAa, DNAc and DNAt are linear chain structure.
The sample solution that described step (3) prepares wherein, is 10 in DNA concentration
-6during M, the concentration of polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer is 10
-6~ 10
-8m can realize the detection to target dna.
Method of the present invention has following features:
(1) DNA of made fluorescence polyelectrolyte and straight chain or hairpin structure is combined by electrostatic interaction and forms molecular probe, now due to base cancellation effect, the fluorescence intensity of pyrene is reduced significantly.
(2) DNA of made fluorescence polyelectrolyte and straight chain or hairpin structure is combined by electrostatic interaction and forms molecular probe, when adding complementary DNA strand in probe solution, fluorescence intensity obviously reduces further, and add not complementary DNA a, DNAc and DNAt strand time, fluorescence intensity changes not quite relatively, thus promptly target dna detected.
The method utilizing non-conjugated fluorescence polyelectrolyte to detect DNA of the present invention is simple, has good selectivity and susceptibility, for new research direction is opened in biological sensing detection field.
Accompanying drawing explanation
Fig. 1 is the synthetic route of example 1 polyacrylic acid pyrene methyl esters of the present invention-poly-(methyl) vinylformic acid decil ester copolymer;
Fig. 2 is polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer
1hNMR;
Sample P (the DMAEMA of Fig. 3 prepared by example 2 of the present invention
+-co-Py), P (DMAEMA
+-co-Py)+DNA1, P (DMAEMA
+-co-Py)+DNA1+DNA3, P (DMAEMA
+-co-Py)+DNA1+DNAa, P (DMAEMA
+-co-Py)+DNA1+DNAc,
P (DMAEMA
+-co-Py)+DNA1+DNAt, correspond to 1 of Fig. 3 (a) (b), 2,3,4,5,6 fluorescence property figure; Sample P (DMAEMA
+-co-Py), P (DMAEMA
+-co-Py)+DNA2, P (DMAEMA
+-co-Py)+DNA2+DNA1, P (DMAEMA
+-co-Py)+DNA2+DNAa, P (DMAEMA
+-co-Py)+DNA2+DNA, P (DMAEMA
+-co-Py)+DNA2+DNAt, correspond to 3(c) 1,2,3,4,5,6 fluorograms of (d).
Fig. 4 is that the present invention detects target dna principle explanatory view.
Fig. 5 is example 3 sample DNA 1+DNA3, P (DMAEMA of the present invention
+-co-Py)+DNA1+DNA3 correspond to 1, the 2 circular dichroism spectrograms of Fig. 5 (a); Sample DNA 2+DNA1, P (DMAEMA
+-co-Py)+DNA2+DNA1 correspond to 1, the 2 circular dichroism spectrograms of Fig. 5 (b).
Sample P (the DMAEMA of Fig. 6 prepared by example 4 of the present invention
+-co-Py), P (DMAEMA
+-co-Py)+DNA1, P (DMAEMA
+-co-Py)+DNA1+DNA3, P (DMAEMA
+-co-Py)+DNA1+DNAa, P (DMAEMA
+-co-Py)+DNA1+DNAc,
P (DMAEMA
+-co-Py)+DNA1+DNAt, correspond to 1 of Fig. 6 (a) (b), 2,3,4,5,6KI cancellation figure; Sample P (DMAEMA
+-co-Py), P (DMAEMA
+-co-Py)+DNA2, P (DMAEMA
+-co-Py)+DNA2+DNA1, P (DMAEMA
+-co-Py)+DNA2+DNAa, P (DMAEMA
+-co-Py)+DNA2+DNAc,
P (DMAEMA
+-co-Py)+DNA2+DNAt, correspond to 1 of Fig. 6 (c) (d), 2,3,4,5,6KI cancellation figure.
Embodiment
Example 1
The tetrahydrofuran solvent after 2ml drying treatment is added in the clean single necked round bottom flask of 25ml, add 0.3mmol multipolymer respectively again, 0.9mmol methyl iodide, at room temperature react 24 hours, after reaction terminates, solvents tetrahydrofurane is filtered out, and then extract product with apparatus,Soxhlet's, wherein extraction agent is tetrahydrofuran (THF), and extraction time is 12 hours, finally again by final fluorescence polyelectrolyte (P (DMAEMA
+-co-Py)) in vacuum drying oven with dry 12 hours of 70 DEG C of temperature.
Example 2
(1) preparation of sample: take appropriate fluorescence polyelectrolyte P (DMAEMA
+-co-Py) be dissolved in damping fluid PBS, make its original concentration be 1 × 10
-5m, goes to dilute the DNA1 that often pipe is equipped with 1od, DNA2 with the damping fluid of 1ml respectively, DNA3, DNAa, DNAc, DNAt, wherein DNA2 is hairpin structure, and DNA1, DNA3, DNAa, DNAc and DNAt are linear chain structure, and DNA2 and DNA1 partial complementarity, with DNAa, DNAc, DNAt is completely not complementary, DNA1 and DNA3 complete complementary, with DNAa, DNAc, DNAt are completely not complementary.Get appropriate fluorescence polyelectrolyte damping fluid and the damping fluid of DNA more respectively, be finally made into P (DMAEMA
+-co-Py), P (DMAEMA
+-co-Py)+DNA1, P (DMAEMA
+-co-Py)+DNA1+DNA3, P (DMAEMA
+-co-Py)+DNA1+DNAa, P (DMAEMA
+-co-Py)+DNA1+DNAc, P (DMAEMA
+-co-Py)+DNA1+DNAt, P (DMAEMA
+-co-Py)+DNA2, P (DMAEMA
+-co-Py)+DNA2+DNA1, P (DMAEMA
+-co-Py)+DNA2+DNAa, P (DMAEMA
+-co-Py)+DNA2+DNAc, P (DMAEMA
+-co-Py)+DNA2+DNAt 11 sample solutions, wherein in each sample solution, the concentration of fluorescence polyelectrolyte keeps 6.0 × 10
-8the concentration of M, DNA keeps 1 × 10
-6m.
(2) sample thermal treatment: the sample prepared in (1) is put into 90 DEG C of drying baker thermal treatments 5 minutes, then moves to rapidly the drying baker thermal treatment half an hour of 40 DEG C.
Finally fluoroscopic examination is carried out to all samples in (2).Fluorescence results as shown in Figure 3.From fluorogram, can find out, made fluorescence polyelectrolyte and the DNA1 of linear chain structure or the DNA2 of hairpin structure are combined by electrostatic interaction and form molecular probe, now due to base cancellation effect, the fluorescence intensity of pyrene are reduced significantly.When to probe solution P (DMAEMA
+-co-Py) add complete complementary DNA3 strand in+DNA1 or to probe solution P (DMAEMA
+-co-Py) when adding partial complementarity DNA1 strand in+DNA2, fluorescence intensity obviously reduces further, and add not complementary DNA a, during DNAc and DNAt strand, fluorescence intensity changes not quite relatively, when this is owing to adding complementary strand, form double-spiral structure, the intercalation of fluorophor pyrene is made to enter in double-strand, as shown in Figure 4, pyrene is made to be in hydrophobic environment, strengthen the cancellation to pyrene fluorescence, and, because intercalation enters double-strand, base cancellation is further strengthened, so when adding complementary strand, the fluorescence intensity obviously cancellation further of pyrene, thus promptly target dna detected.
Example 3
(1) preparation of sample: take appropriate fluorescence polyelectrolyte P (DMAEMA
+-co-Py) be dissolved in damping fluid PBS, make its original concentration be 1 × 10
-5m, go respectively to dilute the DNA1 that often pipe is equipped with 1od, DNA2, DNA3 with the damping fluid of 1ml, wherein DNA2 is hairpin structure, and DNA1, DNA3 are linear chain structure, and DNA2 and DNA1 partial complementarity, DNA1 and DNA3 complete complementary.Get appropriate fluorescence polyelectrolyte damping fluid and the damping fluid of DNA more respectively, be finally made into P (DMAEMA
+-co-Py)+DNA1+DNA3, DNA1+DNA3, P (DMAEMA
+-co-Py)+DNA2+DNA1, a DNA2+DNA14 sample solution, wherein in each sample solution, the concentration of fluorescence polyelectrolyte keeps 4.0 × 10
-6the concentration of M, DNA keeps 1 × 10
-4m.
(2) sample thermal treatment: the sample prepared in (1) is put into 90 DEG C of drying baker thermal treatments 5 minutes, then moves to rapidly the drying baker thermal treatment half an hour of 40 DEG C.
Finally circular dichroism spectrum detection is carried out to all samples in (2), detected result as shown in Figure 5, as can be seen from circular dichroism spectrogram, relative to only containing the DNA1+DNA3 of heteroduplex, DNA2+DNA1 circular dichroism spectrogram, adds fluorescence polyelectrolyte and sample P (DMAEMA
+-co-Py)+DNA1+DNA3, P (DMAEMA
+-co-Py) there is significantly change in the circular dichroism spectrogram of+DNA2+DNA1, if there is electrostatic or gully when acting in fluorescence polyelectrolyte and DNA, be not enough to the circular dichroism spectrogram affecting DNA double chain, only have intercalation to be just enough to duplex structure is rubbed, thus change the circular dichroism spectrogram of DNA double chain structure itself.So, confirm that the intercalation of fluorophor pyrene enters in DNA double chain structure further by circular dichroism spectrogram, thus detect complementary DNA.
Example 4
(1) preparation of sample: take appropriate fluorescence polyelectrolyte P (DMAEMA+-co-Py) and be dissolved in damping fluid PBS, make its original concentration be 1 × 10
-5m, goes to dilute the DNA1 that often pipe is equipped with 1od, DNA2 with the damping fluid of 1ml respectively, DNA3, DNAa, DNAc, DNAt, wherein DNA2 is hairpin structure, and DNA1, DNA3, DNAa, DNAc and DNAt are linear chain structure, and DNA2 and DNA1 partial complementarity, with DNAa, DNAc, DNAt is completely not complementary, DNA1 and DNA3 complete complementary, with DNAa, DNAc, DNAt are completely not complementary.Get appropriate fluorescence polyelectrolyte damping fluid and the damping fluid of DNA more respectively, be finally made into P (DMAEMA
+-co-Py), P (DMAEMA
+-co-Py)+DNA1, P (DMAEMA
+-co-Py)+DNA1+DNA3, P (DMAEMA
+-co-Py)+DNA1+DNAa, P (DMAEMA
+-co-Py)+DNA1+DNAc, P (DMAEMA
+-co-Py)+DNA1+DNAt, P (DMAEMA
+-co-Py)+DNA2, P (DMAEMA
+-co-Py)+DNA2+DNA1, P (DMAEMA
+-co-Py)+DNA2+DNAa, P (DMAEMA
+-co-Py)+DNA2+DNAc, P (DMAEMA
+-co-Py)+DNA2+DNAt11 sample solution, wherein in each sample solution, the concentration of fluorescence polyelectrolyte keeps 6.0 × 10
-8the concentration of M, DNA keeps 1 × 10
-6m.
(2) sample thermal treatment: the sample prepared in (1) is put into 90 DEG C of drying baker thermal treatments 5 minutes, then moves to rapidly the drying baker thermal treatment half an hour of 40 DEG C.
(3) KI quenching experiments: the concentration that in the sample prepared in (2), the prior deionized water of dropping prepares is the KI solution of 0.06M, each dropping 5 μ L, often drip once, survey first order fluorescence, each sample repeats dropping 10 times, thus calculate the cancellation constant of each sample, wherein the calculation formula of cancellation constant is as follows:
I
0/I=1+K
SV[I
-]
Can be drawn by Fig. 6, sample P (DMAEMA
+-co-Py), P (DMAEMA
+-co-Py)+DNA1, P (DMAEMA
+-co-Py)+DNA1+DNA3, P (DMAEMA
+-co-Py)+DNA1+DNAa, P (DMAEMA
+-co-Py)+DNA1+DNAc, P (DMAEMA
+-co-Py) the cancellation constant of+DNA1+DNAt is respectively 1.76 × 10
3, 0.77 × 10
3, 0.55 × 10
3, 0.83 × 10
3, 0.93 × 10
3, and 0.76 × 10
3m
-1, obvious sample P (DMAEMA
+-co-Py) the cancellation constant of+DNA1+DNA3 is minimum, equally, sample P (DMAEMA
+-co-Py)+, P (DMAEMA
+-co-Py)+DNA2, P (DMAEMA
+-co-Py)+DNA2+DNA1, P (DMAEMA
+-co-Py)+DNA2+DNAa, P (DMAEMA
+-co-Py)+DNA2+DNAc, P (DMAEMA
+-co-Py) in+DNA2+DNAt cancellation constant, sample P (DMAEMA
+-co-Py) the cancellation constant of+DNA2+DNA1 is minimum, and this is because fluorophor is intercalated in double-stranded DNA, makes fluorophor pyrene obtain better protecting, avoids I around
-to the cancellation of pyrene, this phenomenon further demonstrate that fluorophor pyrene is by being intercalated in double-stranded DNA.
Claims (4)
1. utilize polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer to detect a method for nucleotide sequence, described method steps comprises:
(1) multipolymer is protonated:
Polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid dimethylamine ethyl ester and methyl iodide are dissolved in organic solvent tetrahydrofuran, react 24 hours at normal temperatures; After reaction terminates, solvents tetrahydrofurane filtered out, and then extract product with apparatus,Soxhlet's, wherein extraction agent is tetrahydrofuran (THF), and extraction time is 12 hours, finally again by fluorescence polyelectrolyte P (DMAEMA
+-co-Py) in vacuum drying oven with dry 12 hours of 70 DEG C of temperature;
(2) preparation of Novel fluorescent molecular probe:
Use buffered soln PBS:10Mm, pH=7.4, original solution is made in polyelectrolyte dilution obtained in (1), equally, buffered soln PBS:10Mm is used respectively, pH=7.4, the single stranded DNA 1 of linear chain structure or the single stranded DNA 2 of hairpin structure are dissolved, finally, then get suitable polyelectrolyte solution and mix with DNA1 or DNA2 solution, by electrostatic interaction capable become the fluorescent molecular probe of novel straight chain or hairpin structure;
(3) preparation of DNA hybridization sample, adopts fluorescent spectrometry and circular dichroism detector to detect associated nucleic acid sequences:
Use buffered soln PBS:10Mm, pH=7.4, dilute not homotactic DNA3, DNAa, DNAc, DNAt; Wherein DNA3 and DNA1 complete complementary pairing, completely not complementary with DNAa, DNAc, DNAt; DNA1 and DNA2 partial complementarity matches, completely not complementary with DNAa, DNAc, DNAt; Get the DNA3 that a certain amount of dissolving is good respectively, in the fluorescent probe sample solution prepared during DNAa, DNAc, DNAt add (2), finally by the thermal treatment of 90 DEG C through 5 minutes of all solution of preparing, then be incubated half an hour at 40 DEG C;
The buffered soln that described step (3) is used is PBS:10Mm, pH=7.4, and DNA used is linear chain structure or hairpin structure: DNA1:5 '-GCA CAT ACATTCTACTTG-3 '; DNA2:5'-GCACAAACAAGTAGAATGTATGTGC-3 '; DNA3:5 '-CGT GTATGT AAG ATG AAC-3 '; DNAa:5 '-AAA AAA AAA AAAAAA AAA-3 '; DNAc:5 '-CCC CCC CCC CCC CCC CCC-3 '; DNAt:5 '-TTT TTT TTTTTT TTT TTT-3 ', wherein DNA2 is hairpin structure, DNA1, DNA3, DNAa, DNAc and DNAt are linear chain structure, and DNA2 and DNA1 partial complementarity, with DNAa, DNAc, DNAt is completely not complementary, DNA1 and DNA3 complete complementary, with DNAa, DNAc, DNAt are completely not complementary.
2. according to method according to claim 1, it is characterized in that: described step (1) polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer contains pyrene fluorophor, makes the polyelectrolyte of synthesis have fluorescence.
3. in accordance with the method for claim 1, it is characterized in that: described step (1) polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer contains water miscible amine groups.
4. in accordance with the method for claim 1, it is characterized in that: described step (1) polyacrylic acid pyrene methyl esters-poly-(methyl) vinylformic acid decil ester copolymer and haloalkane BrCH
3, or ICH
3, or BrCH
2cH
3, or ICH
2cH
3, or BrCH
2cH
2cH
3, or ICH
2cH
2cH
3, or BrCH
2cH
2cH
2cH
3, or ICH
2cH
2cH
2cH
3, be dissolved in organic solvent tetrahydrofuran, multipolymer is protonated, enhance the water-soluble of multipolymer, wherein the ratio 1:1 ~ 1:10 of fluorescent copolymer and the amount of haloalkane.
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| On the binding of Cationic Water-Soluble Conjugated Polymers to DNA: Electrostatic and Hydrophobic Interactions;Fan Xia et al;《NIH Public Access》;20100203;第132卷(第4期);第1－3页 * |
| 荧光聚合物研究进展;武照强等;《化学进展》;20070930;第19卷(第9期);全文 * |
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